EP1179065A2 - Molecules completes exprimees dans des tissus humains - Google Patents

Molecules completes exprimees dans des tissus humains

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Publication number
EP1179065A2
EP1179065A2 EP00935966A EP00935966A EP1179065A2 EP 1179065 A2 EP1179065 A2 EP 1179065A2 EP 00935966 A EP00935966 A EP 00935966A EP 00935966 A EP00935966 A EP 00935966A EP 1179065 A2 EP1179065 A2 EP 1179065A2
Authority
EP
European Patent Office
Prior art keywords
flexht
polynucleotide
polypeptide
sequence
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00935966A
Other languages
German (de)
English (en)
Inventor
Henry Yue
Y. Tom Tang
Preeti Lal
Roopa Reddy
Sajeev Batra
Mariah R. Baughn
Junming Yang
Yalda Azimzai
Dyung Aina M. Lu
Janice Au-Young
Leo L. Shih
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Genomics Inc
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Filing date
Publication date
Application filed by Incyte Genomics Inc filed Critical Incyte Genomics Inc
Publication of EP1179065A2 publication Critical patent/EP1179065A2/fr
Withdrawn legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to nucleic acid and amino acid sequences of full-length molecules expressed in human tissues and to the use of these sequences in the analysis of human gene expression, genetic linkage, and genetic variability, and in the diagnosis, treatment, and prevention of developmental, cell proliferative, and immunological disorders.
  • Cancer Aberrant expression or mutations in genes and their products may cause, or increase susceptibility to, a variety of human diseases such as cancer and other cell proliferative disorders.
  • the identification of these genes and their products is the basis of an ever-expanding effort to finding markers for early detection of diseases and targets for their prevention and treatment.
  • cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body.
  • the development of cancer, or oncogenesis is often correlated with the conversion of a normal gene into a cancer-causing gene, or oncogene, through abnormal expression or mutation.
  • Oncoproteins the products of oncogenes, include a variety of molecules that influence cell proliferation, such as growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins.
  • tumor- suppressor genes are involved in inhibiting cell proliferation. Mutations which reduce or abrogate the function of tumor- suppressor genes result in aberrant cell proliferation and cancer.
  • DNA-based arrays can provide an efficient, high-throughput method to examine gene expression and genetic variability. For example, SNPs. or single nucleotide polymorphisms, are the most common type of human genetic variation.
  • DNA-based arrays can dramatically accelerate the discovery of SNPs in hundreds and even thousands of genes. Likewise, such arrays can be used for SNP genotyping in which DNA samples from individuals or populations are assayed for the presence of selected SNPs. These approaches will ultimately lead to the systematic identification of all genetic variations in the human genome and the correlation of certain genetic variations with disease susceptibility, responsiveness to drug treatments, and other medically relevant information. (See, for example, Wang, D.G. et al. (1998) Science 280:1077-1082.) DNA-based array technology is especially important for the rapid analysis of global gene expression patterns. For example, genetic predisposition, disease, or therapeutic treatment may directly or indirectly affect the expression of a large number of genes in a given tissue.
  • a profile, or transcript image of all the genes that are expressed and the levels at which they are expressed in that particular tissue.
  • a profile generated from an individual or population affected with a certain disease or undergoing a particular therapy may be compared with a profile likewise generated from a control individual or population.
  • Such analysis does not require knowledge of gene function, as the expression profiles can subjected to mathematical analyses which simply treat each gene as a marker.
  • gene expression profiles may help dissect biological pathways by identifying all the genes expressed, for example, at a certain developmental stage, in a particular tissue, or in response to disease or treatment. (See, for example, Lander, E.S. et al. (1996) Science 274:536-539.)
  • the invention features purified polypeptides, full-length molecules expressed in human tissues, referred to collectively as "'FLEXHT “ ' and individually as “FLEXHT-1.
  • FLEXHT-2 purified polypeptides, full-length molecules expressed in human tissues, referred to collectively as "'FLEXHT " ' and individually as “FLEXHT-1.
  • FLEXHT-2 purified polypeptides, full-length molecules expressed in human tissues, referred to collectively as "'FLEXHT " ' and individually as “FLEXHT-1.
  • FLEXHT-2 "FLEXHT-3
  • FLEXHT-4 FLEXHT-5
  • FLEXHT-6 FLEXHT-7.
  • FLEXHT-8 " “FLEXHT-9, "
  • the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 -55.
  • the invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-55.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:56-l 10.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -55 , b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the invention provides a cell transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-55.
  • the invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the pharmaceutical composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional FLEXHT, comprising administering to a patient in need of such treatment the pharmaceutical composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a pharmaceutical composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional FLEXHT, comprising administering to a patient in need of such treatment the pharmaceutical composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from tlie group consisting of SEQ ID NO: 1 -55 , b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • the invention provides a pharmaceutical composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with overexpression of functional FLEXHT, comprising administering to a patient in need of such treatment the pharmaceutical composition.
  • the invention further provides a method of screening for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-55, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -55 , c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-55.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:56-l 10, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
  • the invention further provides an isolated polynucleotide comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence which is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, and b) an RNA equivalent of a).
  • the invention also provides a composition comprising at least one of these isolated polynucleotides and a detectable label, said composition being useful for the detection of altered expression of human FLEXHT.
  • the invention further provides a microarray wherein at least one element of the microa ⁇ ay is an isolated polynucleotide comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence which is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO:56-l 10, and b) an RNA equivalent of a).
  • the invention also provides a method of using the microarray for generating a transcript image of a sample which contains polynucleotides.
  • the method comprises a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
  • Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding FLEXHT.
  • Table 2 shows features of each polypeptide sequence, including protein phosphorylation and glycosylation sites.
  • Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues: and the vector into which each cDNA was cloned.
  • Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding FLEXHT were isolated.
  • Table 5 shows features of selected polypeptide sequences, including the identity of the polypeptide. potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of FLEXHT.
  • Table 6 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
  • FLEXHT refers to the amino acid sequences of substantially purified FLEXHT obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of
  • FLEXHT FLEXHT.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of FLEXHT either by directly interacting with FLEXHT or by acting on components of the biological pathway in which FLEXHT participates.
  • allelic variant is an alternative form of the gene encoding FLEXHT. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • nucleic acid sequences encoding FLEXHT include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as FLEXHT or a polypeptide with at least one functional characteristic of FLEXHT. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding FLEXHT, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding FLEXHT.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent FLEXHT.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of FLEXHT is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of FLEXHT.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of FLEXHT either by directly interacting with FLEXHT or by acting on components of the biological pathway in which FLEXHT participates.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind FLEXHT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • RNA e.g., a mouse, a rat, or a rabbit
  • antigenic determinant '* refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition capable of base-pairing with the "sense "" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA: peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • PNA peptide nucleic acid
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
  • the designation '"negative” or “minus” can refer to the antisense strand, and the designation “positive " ' or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic FLEXHT, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • compositions comprising a given polynucleotide sequence " and a “composition comprising a given amino acid sequence " ' refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding FLEXHT or fragments of FLEXHT may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS). and other components (e.g., Denhardt ' s solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate
  • other components e.g., Denhardt ' s solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence ' refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl. acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • a “fragment” is a unique portion of FLEXHT or the polynucleotide encoding FLEXHT which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ID NO:56-l 10 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:56-l 10, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:56-l 10 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:56-l 10 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO:56-l 10 and the region of SEQ ID NO:56-l 10 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:l-55 is encoded by a fragment of SEQ ID NO:56-l 10.
  • a fragment of SEQ ID NO: 1-55 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:l-55.
  • a fragment of SEQ ID NO:l-55 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO: 1-55.
  • the precise length of a fragment of SEQ ID NO: 1 -55 and the region of SEQ ID NO: 1 -55 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a “full-length "' polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full- length " polynucleotide sequence encodes a "full-length "' polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and "'% identity, " as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • the "weighted " residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences " can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf bl2.html. The "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings.
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity '* and % identity, " as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • NCBI BLAST software suite may be used.
  • Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40. at least 50. at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing " step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS. and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T--) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response * ' can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines. and other signaling molecules, which may affect cellular and systemic defense systems.
  • factors e.g., cytokines, chemokines. and other signaling molecules, which may affect cellular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of FLEXHT which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment "” also includes any polypeptide or oligopeptide fragment of FLEXHT which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • array element refers to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of FLEXHT.
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of FLEXHT.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • Post-translational modification of an FLEXHT may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of FLEXHT.
  • Probe refers to nucleic acid sequences encoding FLEXHT, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primarymers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60. 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0 5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA)
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose
  • OLIGO 4 06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases
  • Similar primer selection programs have incorporated additional features for expanded capabilities For example, the P ⁇ mOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center.
  • the P ⁇ mer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "misp ⁇ mmg library," which sequences to avoid as primer binding sites are user-specified P ⁇ mer3 is useful, in particular, for the selection of oligonucleotides for microarrays (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user s specific needs )
  • the P ⁇ meGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments The oligonu
  • a "recombinant nucleic acid " ' is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g.. by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a "regulatory element "" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules ' are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope “A. " the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants, and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a “variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 857c, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "allelic " (as defined above), “splice,” “species, “ or “polymorphic " variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymorphisms
  • the presence of SNPs may be indicative of. for example, a certain population, a disease state, or a propensity for a disease state.
  • a “variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new full-length molecules expressed in human tissues (FLEXHT), the polynucleotides encoding FLEXHT, and the use of these compositions for the analysis of human gene expression, genetic linkage, and genetic variability, and for the diagnosis, treatment, or prevention of developmental, cell proliferative, and immunological disorders.
  • Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding
  • FLEXHT Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively.
  • Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each FLEXHT were identified, and column 4 shows the cDNA libraries from which these clones were isolated.
  • Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each FLEXHT and are useful as fragments in hybridization technologies.
  • column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; and column 4 shows potential glycosylation sites.
  • the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding FLEXHT.
  • the first column of Table 3 lists the nucleotide SEQ ID NOs.
  • Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO:56-l 10 and to distinguish between SEQ ID NO:56-l 10 and related polynucleotide sequences.
  • polypeptides encoded by the indicated fragments of SEQ ID NO:56, SEQ ID NO:58-69, SEQ ID NO.71-72, SEQ ID NO:74-76, SEQ ID NO:79-85, SEQ ID NO:87, SEQ ID NO:90-98, SEQ ID NO:100-103, and SEQ ID NO:105-110 are useful, for example, as immunogenic peptides.
  • Column 3 lists tissue categories which express FLEXHT as a fraction of total tissues expressing FLEXHT.
  • Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding FLEXHT were isolated.
  • Column 1 references the nucleotide SEQ ID NOs
  • column 2 shows the cDNA libraries from which these clones were isolated
  • column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
  • column 1 references the SEQ ID NO
  • column 2 shows the identity of the polypeptide along with relevant citations, all of which are expressly incorporated by reference herein in their entirety
  • column 3 shows the amino acid residues comprising signature sequences, domains, and motifs, and homologous sequences as identified by BLAST analysis
  • column 4 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 4 were used to characterize each polypeptide through sequence homology and protein motifs.
  • the invention incorporates the nucleic acid sequences disclosed in the Sequence Listing and the use of these sequences in the diagnosis and treatment of disease states characterized by altered expression of FLEXHT genes or defects in FLEXHT protein function.
  • the invention further utilizes these sequences in hybridization and amplification technologies, and in particular, in technologies which comprehensively assess gene expression patterns correlated with specific cells or tissues and their responses in vivo or in vitro to pharmaceutical agents, toxins, and other treatments.
  • the sequences of the present invention are used to develop a transcript image for a particular cell or tissue.
  • the invention also encompasses FLEXHT variants.
  • a preferred FLEXHT variant is one which has at least about 80%, or alternatively at least about 90%. or even at least about 95% amino acid sequence identity to the FLEXHT amino acid sequence, and which contains at least one functional or structural characteristic of FLEXHT
  • the invention also encompasses polynucleotides which encode FLEXHT
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO 56-110 which encodes FLEXHT
  • the polynucleotide sequences of SEQ ID NO 56-110 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ⁇ bose instead of deoxyribose
  • the invention also encompasses a variant of a polynucleotide sequence encoding FLEXHT
  • a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding FLEXHT
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO 56-110 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO 56-110
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of FLEXHT
  • nucleotide sequences which encode FLEXHT and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring FLEXHT under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding FLEXHT or its derivatives possessing a substantially different codon usage, e g , inclusion of non-naturally occurring codons Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • Other reasons for substantially altering the nucleotide sequence encoding FLEXHT and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence
  • the invention also encompasses production of DNA sequences which encode FLEXHT and FLEXHT derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding FLEXHT or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:56-l 10 and fragments thereof under various conditions of stringency.
  • SEQ ID NO:56-l 10 and fragments thereof under various conditions of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g.. formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25 °C, more preferably of at least about 42°C, and most preferably of at least about 68°C.
  • wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Further examples of hybridization conditioas, including annealing and wash conditions, are described in "Definitions.” cDNA Sequencing
  • Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH).
  • Taq polymerase Per kin-Elmer
  • thermostable T7 polymerase Amersham Pharmacia Biotech, Piscataway NJ
  • combinations of polymer ases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies. Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Perkin- Elmer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
  • the nucleic acid sequences encoding FLEXHT may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector.
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g...
  • primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68 °C to 72°C.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode FLEXHT may be cloned in recombinant DNA molecules that direct expression of FLEXHT, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express FLEXHT.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter FLEXHT-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of FLEXHT, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. e
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • sequences encoding FLEXHT may be synthesized, in whole or in part, using chemical methods well known in the art.
  • chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • FLEXHT itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or solid-phase techniques.
  • Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Per kin-Elmer). Additionally, the amino acid sequence of FLEXHT, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See. e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences encoding FLEXHT or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5 ' and 3 ' untranslated regions in the vector and in polynucleotide sequences encoding FLEXHT. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding FLEXHT. Such signals include the ATG initiation codon and adjacent sequences, e.g.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding FLEXHT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g.. baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g.. baculovirus)
  • plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV, or
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
  • the invention is not limited by the host cell employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding FLEXHT.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding FLEXHT can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies).
  • Yeast expression systems may be used for production of FLEXHT.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • PGH promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or inttacellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See. e.g., Ausubel, 1995, supra; Bitter, supra; and Scorer, supra.)
  • Plant systems may also be used for expression of FLEXHT. Transcription of sequences encoding FLEXHT may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See. e.g., Coruzzi, supra: Broglie, supra; and Winter, supra.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.)
  • a number of viral-based expression systems may be utilized.
  • sequences encoding FLEXHT may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses FLEXHT in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of
  • DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g.. Harrington. J.J. et al. (1997) Nat. Genet. 15:345-355.)
  • stable expression of FLEXHT in cell lines is preferred.
  • sequences encoding FLEXHT can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ' and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and /.at confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and isD. which alter cellular requirements for metabolites.
  • Visible markers e.g.. anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants. but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding FLEXHT is inserted within a marker gene sequence
  • transformed cells containing sequences encoding FLEXHT can be identified by the absence of marker gene function
  • a marker gene can be placed in tandem with a sequence encoding FLEXHT under the control of a single promoter Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well
  • host cells that contain the nucleic acid sequence encoding FLEXHT and that express FLEXHT may be identified by a variety of procedures known to those of skill in the art These procedures include, but are not limited to. DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or lmmunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences
  • Immunological methods for detecting and measuring the expression of FLEXHT using either specific polyclonal or monoclonal antibodies are known in the art Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs). and fluorescence activated cell sorting (FACS)
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • Host cells transformed with nucleotide sequences encoding FLEXHT may be cultured under conditions suitable for the expression and recovery of the protein from cell culture
  • the protein produced by a transformed cell may be secreted or retained lntracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode FLEXHT may be designed to contain signal sequences which direct secretion of FLEXHT through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro " form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK. HEK293, and WI38) are available from the American Type Culture
  • ATCC Manassas VA
  • UCC Manassas VA
  • natural, modified, or recombinant nucleic acid sequences encoding FLEXHT may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric FLEXHT protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of FLEXHT activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c- myc. and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the FLEXHT encoding sequence and the heterologous protein sequence, so that FLEXHT may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995. supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled FLEXHT may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, ⁇ S-methionine. Fragments of FLEXHT may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques (See, e g .
  • Protein synthesis may be performed by manual techniques or by automation Automated synthesis may be achieved for example, using the ABI 431 A peptide synthesizer (Perkin-Elmer) Various fragments of FLEXHT may be synthesized separately and then combined to produce the full length molecule Screening Assays
  • FLEXHT of the present invention or fragments thereof may be used to screen for compounds that specifically bind to FLEXHT At least one and up to a plurality of test compounds may be screened lor specific binding to FLEXHT Examples of test compounds include antibodies, oligonucleotides, proteins (e g , receptors), or small molecules
  • the compound thus identified is closely related to the natural gand of FLEXHT, e g , a hgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (See, Cohgan, J E et al (1991) Current Protocols in Immunology 1(2) Chapter 5 )
  • the compound can be closely related to the natural receptor to which FLEXHT binds, or to at least a fragment of the receptor, e g , the hgand binding site
  • the compound can be rationally designed using known techniques
  • screemng for these compounds involves producing approp ⁇ ate cells which express FLEXHT, either as a secreted protein or on the cell membrane
  • Preferred cells include cells from mammals, yeast, Drosophila, or E_ coh Cells expressing FLEXHT or cell membrane fractions which contain FLEXHT are then contacted with a test compound and binding, stimulation, or inhibition of activity of either FLEXHT or the compound is
  • an assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope enzyme conjugate, or other detectable label
  • the assay may comprise the steps of combimng at least one test compound with FLEXHT, either m solution or affixed to a solid support, and detecting the binding of FLEXHT to the compound
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor
  • the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support FLEXHT of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of FLEXHT
  • Such compounds may include agonists, antagonists, or partial or inverse agomsts
  • an assay is performed under conditions permissive for FLEXHT activity, wherein FLEXHT is combined with at least one
  • an ELISA assay using, e g . a monoclonal or polyclonal antibody can measure polypeptide level in a sample
  • the antibody can measure polypeptide level by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate
  • the molecules discovered using these assays can be used to treat disease or to b ⁇ ng about a particular result in a patient (e g , blood vessel growth) by activating or inhibiting the polypeptide/molecule Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues
  • Transc ⁇ pt Imaging Another embodiment relates to the use of polynucleotide sequences encoding FLEXGEM to develop a transcript image of a tissue or cell type
  • a transc ⁇ pt image is the collective pattern of gene expression by a particular tissue or cell type under given conditions and at a given time This pattern of gene expression is defined by the number of expressed genes and their abundance
  • the polynucleotide sequences of the present invention may be used to develop a transc ⁇ pt image of a tissue or cell type by hybridizing, preferably in a microarray format, the polynucleotide sequences of the present invention
  • Transcript images which profile FLEXGEM gene expression may be generated using transc ⁇ pts isolated from tissues, cell lines, biopsies, or other biological samples
  • the transc ⁇ pt image may thus reflect FLEXGEM gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line
  • Transcript images may be used to profile FLEXGEM gene expression in distinct tissue types This process can be used to determine FLEXGEM gene activity in a particular tissue type relative to this activity in a different tissue type
  • Transc ⁇ pt images may be used to generate a profile of FLEXGEM gene expression characteristic of diseased tissue
  • Transc ⁇ pt images of tissues before and after treatment may be used for diagnostic purposes, to momtor the progression of disease, and to momtor the efficacy of drug treatments for diseases which affect the activity of genes encoding FLEXGEM
  • Transcript images which profile FLEXGEM gene expression may also be used in conjunction with in vitro model systems and prechnical evaluation of pharmaceuticals
  • Transcript images of cell lines can be used to assess FLEXGEM activity and/or to identify cell lines that lack or misregulate this activity. Such cell lines may then be treated with pharmaceutical agents, and a transcript image following treatment may indicate the efficacy of these agents in restoring desired levels of this activity.
  • a similar approach may be used to assess the toxicity of pharmaceutical agents as reflected by undesirable changes in FLEXGEM activity.
  • Candidate pharmaceutical agents may be evaluated by comparing their associated transcript images with those of pharmaceutical agents of known effectiveness.
  • polynucleotides encoding FLEXHT or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
  • ES embryonic stem
  • Such techniques are well known in the art and are useful for the generation of animal models of human disease.
  • mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo: Capecchi, M.R. (1989) Science 244:1288-1292).
  • a marker gene e.g., the neomycin phosphotransferase gene (neo: Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transfe ⁇ ed to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding FLEXHT may also be manipulated in vitro in ES cells derived from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm. and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding FLEXHT can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding FLEXHT is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress FLEXHT e.g., by secreting FLEXHT in its milk, may also serve as a convenient source of that protein (Janne. J. et al. (1998) Biotechnol. Annu. Rev. 4:55-
  • FLEXHT Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of FLEXHT and full-length molecules expressed in human tissues.
  • the expression of FLEXHT is closely associated with cell proliferation and the immune response. Therefore, FLEXHT appears to play a role in developmental, cell proliferative, and immunological disorders. In the treatment of disorders associated with increased FLEXHT expression or activity, it is desirable to decrease the expression or activity of FLEXHT. In the treatment of disorders associated with decreased FLEXHT expression or activity, it is desirable to increase the expression or activity of FLEXHT.
  • FLEXHT or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of FLEXHT.
  • disorders include, but are not limited to, a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms " tumor, aniridia.
  • Smith-Magenis syndrome myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, ci ⁇ hosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancer
  • MCTD mixed connective tissue disease
  • melanoma myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and an immunological disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison " s disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, ci ⁇ hosis, contact dermatitis.
  • an immunological disorder such
  • MCTD mixed connective tissue disease
  • multiple sclerosis myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis.
  • osteoporosis pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter ' s syndrome, rheumatoid arthritis, scleroderma, Sjogren ' s syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma.
  • a vector capable of expressing FLEXHT or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of FLEXHT including, but not limited to, those described above.
  • a pharmaceutical composition comprising a substantially purified FLEXHT in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of FLEXHT including, but not limited to, those provided above.
  • an agonist which modulates the activity of FLEXHT may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of FLEXHT including, but not limited to, those listed above.
  • an antagonist of FLEXHT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of FLEXHT.
  • disorders include, but are not limited to, those developmental, cell proliferative, and immunological disorders described above.
  • an antibody which specifically binds FLEXHT may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express FLEXHT.
  • a vector expressing the complement of the polynucleotide encoding FLEXHT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of FLEXHT including, but not limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of FLEXHT may be produced using methods which are generally known in the art.
  • purified FLEXHT may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind FLEXHT.
  • Antibodies to FLEXHT may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies.
  • Neutralizing antibodies i.e., those which inhibit dimer formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with FLEXHT or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund ' s, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols. polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to FLEXHT have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of FLEXHT amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced. Monoclonal antibodies to FLEXHT may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • hybridoma technique examples include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton. D.R. (1991) Proc. Natl. Acad. Sci.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (See, e g , Orlandi, R et al (1989) Proc Natl Acad Sci USA 86 3833-3837, Winter, G et al (1991) Nature 349 293-299 ) Antibody fragments which contain specific binding sites for FLEXHT may also be generated
  • fragments include, but are not limited to, F(ab " ) 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab * )2 fragments
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (See, e g , Huse, W D et al (1989) Science 246 1275-1281 )
  • lmmunoassays may be used for screening to identify antibodies having the desired specificity
  • Numerous protocols for competitive binding or lmmunoradiomet ⁇ c assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art
  • Such lmmunoassays typically involve the measurement of complex formation between FLEXHT and its specific antibody
  • a two-site, monoclonal-based lmmunoassay utihzing monoclonal antibodies reactive to two non-interfering FLEXHT epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra)
  • K association constant
  • the I determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple FLEXHT epitopes, represents the average affinity, or avidity, of the antibodies for FLEXHT
  • the I determined for a preparation of monoclonal antibodies, which are monospecific for a particular FLEXHT epitope, represents a true measure of affinity High-affinity antibody preparations with F , ranging from about 10 9 to 10 12 L/mole are prefe ⁇ ed for use in lmmunoassays in which the FLEXHT- antibody complex must withstand rigorous manipulations
  • Low-affinity antibody preparations with K ranging from about 10 6 to 10 7 L/mole are prefe ⁇ ed for use in lmmunopurification and similar procedures which ultimately require dissociation of FLEXHT, preferably in active form, from the antibody (Catty, D (1988) Antibodies Volume I A Practical Approach, IRL Press, Washington DC, Liddell, J E and A Cryer (1991)
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of FLEXHT-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (See, e.g , Catty, supra, and Coligan et al , supra )
  • the polynucleotides encoding FLEXHT may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding FLEXHT
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
  • antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding FLEXHT (See, e.g , Agrawal, S., ed (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.)
  • Antisense sequences can be delivered mtracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (See, e.g., Slater, J.E. et al. (1998) J. Allergy Chn. Immunol. 102(3)'469-475, and Scanlon, K.J. et al.
  • Antisense sequences can also be introduced mtracellularly through the use of viral vectors, such as rettovirus and adeno-associated virus vectors (See, e g., Miller, A.D (1990) Blood 76.271, Ausubel, supra, Uckert, W. and W Walther (1994) Pharmacol. Ther. 63(3).323-347 )
  • viral vectors such as rettovirus and adeno-associated virus vectors
  • Other gene delivery mechanisms include hposome-de ⁇ ved systems, artificial viral envelopes, and other systems known in the art.
  • Rossi, J.J. (1995) Br Med. Bull 51(l).217-225, Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11).1308-1315, and Morns, M C et al. (1997) Nucleic Acids Res 25(14).2730-2736.)
  • polynucleotides encoding FLEXHT may be used for somatic or germline gene therapy
  • Gene therapy may be performed to (I) co ⁇ ect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288.669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M et al. (1995) Science 270.475-480, Bordignon, C et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • a protein which affords protection against intracellular parasites e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi).
  • human retroviruses such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399)
  • HBV human immunodeficiency virus
  • FLEXHT FLEXHT
  • the expression of FLEXHT from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
  • FLEXHT are treated by constructing mammalian expression vectors encoding FLEXHT and introducing these vectors by mechanical means into FLEXHT-deficient cells.
  • Mechanical ttansfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene ttansfer, and (v) the use of DNA ttansposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91 :501-510; Boulay, J-L. and H Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
  • Expression vectors that may be effective for the expression of FLEXHT include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invittogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Sttatagene, La Jolla CA), and PTET-OFF,
  • FLEXHT may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551 ; Gossen, M. et al.
  • CMV cytomegalovirus
  • RSV40 virus SV40 virus
  • TK thymidine kinase
  • ⁇ -actin genes e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-55
  • liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invittogen
  • PERFECT LIPID TRANSFECTION KIT available from Invittogen
  • transformation is performed using the calcium phosphate method (Graham. F.L. and A.J. Eb (1973) Virology 52:456-467). or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to FLEXHT expression are treated by constructing a rettovirus vector consisting of (i) the polynucleotide encoding FLEXHT under the control of an independent promoter or the rettovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional rettovirus ⁇ ' s-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Rettovirus vectors e.g., PFB and PFBNEO
  • PFB and PFBNEO are commercially available (Sttatagene) and are based on published data (Riviere, I. et al.
  • the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a ttopism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61 :1647-1650; Bender, M.A. et al. (1987) J. Virol. 61 :1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
  • VSVg vector producing cell line
  • U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining rettovirus packaging cell lines and is hereby incorporated by reference. Propagation of rettovirus vectors, ttansduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
  • an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding FLEXHT to cells which have one or more genetic abnormalities with respect to the expression of FLEXHT.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S.
  • Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy " ), hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutt. 19:511-544; and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
  • a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding FLEXHT to target cells which have one or more genetic abnormalities with respect to the expression of FLEXHT
  • the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing FLEXHT to cells of the central nervous system, lor which HSV has a tropism
  • the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art
  • a replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X et al (1999) Exp Eye Res 169 385-395)
  • the construction of a HSV-1 virus vector has also been disclosed in detail in U S Patent Number 5,804,413 to DeLuca ( * Herpes simplex virus strains for gene ttansfer '), which is hereby incorporated by reference U S Patent Number 5
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding FLEXHT to target cells
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • a subgenomic RNA is generated that normally encodes the viral capsid proteins
  • This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e g , protease and polymerase)
  • inserting the coding sequence for FLEXHT into the alphavirus genome in place of the capsid-coding region results in the production of
  • Oligonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee. J.E. et al. (1994) in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block ttanslation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding FLEXHT.
  • RNA sequences within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC
  • short RNA sequences of between 15 and 20 ribonucleotides, co ⁇ esponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding FLEXHT. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA. constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase inttacellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 ' and/or 3' ends of the molecule, or the use of phosphorothioate or 2 " O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding FLEXHT.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non- macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specifically inhibits expression of the polynucleotide encoding FLEXHT may be therapeutically useful, and in the treament of disorders associated with decreased FLEXHT expression or activity, a compound which specifically promotes expression of the polynucleotide encoding FLEXHT may be therapeutically useful.
  • test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occu ⁇ ing or non- natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
  • a sample comprising a polynucleotide encoding FLEXHT is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding FLEXHT are assayed by any method commonly known in the art.
  • the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding FLEXHT.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435: Arndt, G.M. et al. (2000) Nucleic Acids Res. 28.E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022.691).
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by ttansfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g.. Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • Such pharmaceutical compositions may consist of FLEXHT, antibodies to FLEXHT, and mimetics, agonists, antagonists, or inhibitors of FLEXHT.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, inttathecal, intraventticular, pulmonary, ttansdermal, subcutaneous, inttaperitoneal, intranasal. enteral, topical, sublingual, or rectal means.
  • compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see. e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers. Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
  • Specialized forms of pharmaceutical compositions may be prepared for direct intracellular delivery of macromolecules comprising FLEXHT or fragments thereof.
  • liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
  • FLEXHT or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are prefe ⁇ ed.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind FLEXHT may be used for the diagnosis of disorders characterized by expression of FLEXHT, or in assays to monitor patients being treated with FLEXHT or agonists, antagonists, or inhibitors of FLEXHT.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for FLEXHT include methods which utilize the antibody and a label to detect FLEXHT in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • FLEXHT FLEXHT
  • ELISAs RIAs
  • FACS FACS-activated FLEXHT
  • body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects
  • the amount of standard complex formation may be quantitated by various methods, such as photomettic means.
  • Quantities of FLEXHT expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding FLEXHT may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of FLEXHT may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of FLEXHT, and to monitor regulation of FLEXHT levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding FLEXHT or closely related molecules may be used to identify nucleic acid sequences which encode FLEXHT.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding FLEXHT, allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the FLEXHT encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:56-l 10 or from genomic sequences including promoters, enhancers, and introns of the FLEXHT gene.
  • Means for producing specific hybridization probes for DNAs encoding FLEXHT include the cloning of polynucleotide sequences encoding FLEXHT or FLEXHT derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ,2 P or 33 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding FLEXHT may be used for the diagnosis of disorders associated with expression of FLEXHT.
  • disorders include, but are not limited to, a developmental disorder such as renal tubular acidosis, anemia, Cushing ' s syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms ' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma
  • arteriosclerosis arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus: and an immunological disorder such as inflammation, actinic keratosis,
  • Graves ' disease Hashimoto ' s thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, nritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis.
  • osteoporosis pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter " s syndrome, rheumatoid arthritis, scleroderma, Sjogren " s syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and exttacorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma.
  • the polynucleotide sequences encoding FLEXHT may be used in Southern or northern analysis, dot blot, or other membrane-based technologies: in PCR technologies: in dipstick, pin, and multiformat ELISA-like assays; and in microa ⁇ ays utilizing fluids or tissues from patients to detect altered FLEXHT expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding FLEXHT may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding FLEXHT may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding FLEXHT in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding FLEXHT, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal sub)ect
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months
  • oligonucleotides designed from the sequences encoding FLEXHT may involve the use of PCR These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro Oligomers will preferably contain a fragment of a polynucleotide encoding FLEXHT, or a fragment of a polynucleotide complementary to the polynucleotide encoding FLEXHT, and will be employed under optimized conditions for identification of a specific gene or condition Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequence
  • oligonucleotide primers derived from the polynucleotide sequences encoding FLEXHT may be used to detect single nucleotide polymorphisms (SNPs) SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans
  • SNP detection include, but are not limited to, smgle-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods
  • SSCP smgle-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from the polynucleotide sequences encoding FLEXHT are used to amplify DNA using the polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like SNPs in the DNA cause differences in the secondary and tert
  • FLEXHT FLEXHT
  • Methods which may also be used to quantify the expression of FLEXHT include radiolabeling or biotinylating nucleotides. coamplification of a control nucleic acid, and interpolating results from standard curves.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • the polynucleotides are also useful for identifying individuals on the basis of minute biological samples, for example, by matching the restriction fragment length polymorphism (RFLP) pattern of a sample ' s DNA to that of an individual ' s DNA.
  • the polynucleotides of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, an individual can be identified through a unique set of DNA sequences. Once a unique ID database is established for an individual, positive identification of that individual can be made from extremely small tissue samples.
  • RFLP restriction fragment length polymorphism
  • DNA-based identification techniques are critical in forensic technology.
  • DNA sequences taken from very small biological samples such as tissues, e.g., hair, skin, or body fluids (e.g., blood, saliva, semen, etc.), can be amplified using, e.g., PCR, to identify individuals.
  • PCR PCR
  • polynucleotides of the present invention can be used as polymorphic markers.
  • reagents capable of identifying the source of a particular tissue.
  • Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
  • polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a particular cell type, in the creation of subtracted cDNA libraries which aid in the discovery of novel polynucleotides, in selection and synthesis of oligomers for attachment to an array or other support, and as an antigen to elicit an immune response.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microa ⁇ ay can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al.. "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference.
  • the microa ⁇ ay may also be used to identify genetic variants, mutations, and polymorphisms.
  • This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • antibodies specific for FLEXHT. or FLEXHT or fragments thereof may be used as elements on a microa ⁇ ay.
  • the microarray may be used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • Microa ⁇ ays may be prepared, used, and analyzed using methods known in the art.
  • nucleic acid sequences encoding FLEXHT may be used to generate hybridization probes useful in mapping the naturally occu ⁇ ing genomic sequence.
  • Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDN A libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDN A libraries.
  • nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which co ⁇ elate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization may be correlated with other physical and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Co ⁇ elation between the location of the gene encoding FLEXHT on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, ca ⁇ ier, or affected individuals.
  • FLEXHT its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located mtracellularly. The formation of binding complexes between FLEXHT and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with FLEXHT, or fragments thereof, and washed. Bound FLEXHT is then detected by methods well known in the art. Purified FLEXHT can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • nucleotide sequences which encode FLEXHT may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cu ⁇ ently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • RNA was purchased from Clontech or isolated from tissues described in Table 4. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or exttacted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stratagene was provided with RNA and constructed the co ⁇ esponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997. supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPH AROSE CL2B, or SEPH AROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), pcDNA2.1 plasmid (Invittogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
  • PBLUESCRIPT plasmid e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), pcDNA2.1 plasmid (Invittogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics,
  • Recombinant plasmids were ttansformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ . DH10B. or ElectroMAX DH10B from Life Technologies.
  • Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
  • a Magic or WIZARD Minipreps DNA purification system Promega
  • an AGTC Miniprep purification kit Edge Biosystems, Gaithersburg MD
  • plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384- well plates, and the concentration of amplified plasmid DNA was quantified fluoromettically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid ttansfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
  • Table 6 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters.
  • the first column of Table 6 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences)
  • Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASER GENE software (DNASTAR) Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences
  • polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis
  • the sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e g , Sambrook, supra, ch 7, Ausubel, 1995, supra, ch 4 and 16 )
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quality in a BLAST alignment.
  • a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared.
  • a product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other.
  • a product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • the results of northern analyses are reported as a percentage distribution of libraries in which the ttanscript encoding FLEXHT occu ⁇ ed.
  • Analysis involved the categorization of cDNA libraries by organ tissue and disease.
  • the organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic.
  • the disease condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
  • the full length nucleic acid sequences of SEQ ID NO:56-l 10 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5 ' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (A ersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min: Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ were as follows: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 1
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electtophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, ttansfe ⁇ ed to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WT), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and ttansfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
  • the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 °C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
  • polynucleotide sequences of SEQ ID NO:56-l 10 are used to obtain 5' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library.
  • Hybridization probes derived from SEQ ID NO:56-l 10 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dexttan bead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transfe ⁇ ed to nylon membranes (Nyttan Plus, Schleicher & Schuell, Durham NH). Hybridization is ca ⁇ ied out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium cittate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
  • Transcript images are generated as described in Seilhamer, J.J. et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference.
  • the linkage or synthesis of array elements upon a microa ⁇ ay can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999). supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to a ⁇ ange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon. D. et al. (1996) Genome Res. 6:639-645; Marshall. A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectromefry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microa ⁇ ay may be assessed. In one embodiment, microa ⁇ ay preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-ttanscriptase, 0.05 pg/ ⁇ l oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • Sequences of the present invention are used to generate array elements.
  • Each a ⁇ ay element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • a ⁇ ay elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia
  • Purified a ⁇ ay elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4% hydrofluoric acid (VWR
  • a ⁇ ay elements are applied to the coated glass substrate using a procedure described in US Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the a ⁇ ay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of a ⁇ ay element sample per slide.
  • Microa ⁇ ays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
  • Microa ⁇ ays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microa ⁇ ays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 °C followed by washes in
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65 °C for 5 minutes and is ali quoted onto the microa ⁇ ay surface and covered with an 1.8 cm 2 coverslip.
  • the a ⁇ ays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of
  • Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 n for excitation of Cy5.
  • the excitation laser light is focused on the a ⁇ ay using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the slide containing the a ⁇ ay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers. In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
  • Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) co ⁇ esponding to the two fluorophores.
  • PMT R1477 Hamamatsu Photonics Systems, Bridgewater NJ
  • Appropriate filters positioned between the a ⁇ ay and the photomultiplier tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each a ⁇ ay is typically scanned twice, one scan per fiuorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the a ⁇ ay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be co ⁇ elated with a weight ratio of hybridizing species of 1 : 100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital
  • AID Analog Devices, Inc., Norwood MA
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fiuorophore' s emission spectrum.
  • Sequences complementary to the FLEXHT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occu ⁇ ing FLEXHT.
  • oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of FLEXHT.
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the FLEXHT-encoding transcript.
  • FLEXHT FLEXHT expression and purification of FLEXHT is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are ttansformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express FLEXHT upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • FLEXHT FLEXHT in eukaryotic cells
  • baculovirus recombinant Autographica californica nuclear polyhedrosis virus
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding FLEXHT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • FLEXHT is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • FLAG an 8-amino acid peptide
  • 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified FLEXHT obtained by these methods can be used directly in the assays in Examples XII and XIV. XL Functional Assays
  • FLEXHT function is assessed by expressing the sequences encoding FLEXHT at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
  • recombinant vector contains the cytomegalovirus promoter.
  • 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death.
  • FLEXHT The influence of FLEXHT on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding FLEXHT and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding FLEXHT and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques.
  • FLEXHT amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995. supra, ch. 11.)
  • oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Perkin-Elmer) using FMOC chemistry and coupled to KLH (Sigma- Aldrich. St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • ABI 431 A peptide synthesizer Perkin-Elmer
  • KLH Sigma- Aldrich. St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund ' s adjuvant.
  • Resulting antisera are tested for antipeptide and anti-FLEXHT activity by, for example, binding the peptide or FLEXHT to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant FLEXHT is substantially purified by immunoaffinity chromatography using antibodies specific for FLEXHT.
  • An immunoaffinity column is consttucted by covalently coupling anti-FLEXHT antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer ' s instructions.
  • FLEXHT Media containing FLEXHT are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of FLEXHT (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/FLEXHT binding (e.g., a buffer of pH 2 to pH 3. or a high concentration of a chaotrope, such as urea or thiocyanate ion), and FLEXHT is collected.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled FLEXHT, washed, and any wells with labeled FLEXHT complex are assayed. Data obtained using different concentrations of FLEXHT are used to calculate values for the number, affinity, and association of FLEXHT with the candidate molecules.
  • molecules interacting with FLEXHT are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989. Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • FLEXHT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • ABI AutoAssembler A program that assembles nucleic acid sequences Perkin-Elmer Applied Biosystems, Foster City, CA
  • HMMER An algorithm for searching a query sequence against Krogh, A et al ( 1994) J Mol Biol , hidden Markov model (HMM)-based databases of protein 235 1501-1531 , Sonnhammer, E L L et al family consensus sequences, such as PFAM (1988) Nucleic Acids Res 26 320 322 families
  • Motifs A piogiam that searches amino acid sequences for patterns Bairoch et al supia, Wisconsin that matched those defined in Prosite Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI

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Abstract

L'invention se rapporte à des molécules complètes exprimées dans des tissus humains, désignées par le terme FLEXHT, et à des polynucléotides qui identifient et codent pour FLEXHT. L'invention se rapporte également à des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention se rapporte également à des méthodes permettant d'analyser l'expression de gènes humains, les relations génétiques et la variabilité génétique, et de diagnostiquer, traiter ou prévenir des troubles associés à l'expression de FLEXHT.
EP00935966A 1999-05-14 2000-05-12 Molecules completes exprimees dans des tissus humains Withdrawn EP1179065A2 (fr)

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AU2002221841A1 (en) * 2000-11-14 2002-05-27 Bayer Aktiengesellschaft Polynucleotide and polypeptide sequences of human purple acid phosphate
WO2002040668A2 (fr) * 2000-11-15 2002-05-23 Apoxis Sa Proteines et sequences d'adn sous-jacentes a ces proteines, utilisees pour traiter les inflammations
US20030219739A1 (en) * 2001-01-30 2003-11-27 Glass David J. Novel nucleic acid and polypeptide molecules
US20020187522A1 (en) * 2001-03-06 2002-12-12 Yan Luo DNA encoding human Cid1
MXPA04003155A (es) * 2001-10-03 2004-07-08 Inmunex Corp Polipeptidos de citoquina de murino y humano.
EP2006687A1 (fr) * 2007-06-20 2008-12-24 Commissariat A L'energie Atomique Procédé de test dýun sujet dont on pense quýil a ou quýil est prédisposé à de lýasthme, des allergies, une maladie atopique ou une sensibilisation atopique

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WO1995014772A1 (fr) * 1993-11-12 1995-06-01 Kenichi Matsubara Signature genique
EP1053319A2 (fr) * 1998-01-28 2000-11-22 Chiron Corporation Genes humains et expression de produits genetiques ii
US6476207B1 (en) * 1998-06-11 2002-11-05 Chiron Corporation Genes and gene expression products that are differentially regulated in prostate cancer

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